The reproductive system of model species such as the domestic cat and its non-domestic relatives offers an unusual opportunity for understanding the changes and adaptations of genes that mediate species isolation and survival. The Laboratory of Genomic Diversity (LGD) has undertaken a comparative physiology approach to describe the aspects of feline reproduction that discriminate between species and allow for behavioral co-adaptation. In addition, empirical methods to develop cryopreservation have been assessed to optimize assisted reproductive technologies in these species, including artificial insemination, in vitro fertilization (IVF) and embryo transfer between these closely related species. Models of how to use new technologies to assess reproductive fitness are emerging to help insure gene diversity and propagate endangered species. Non-invasive hormone metabolite monitoring assays, artificial insemination techniques and genome resource banking have been developed to aid in studies examining the adaptive differences among the Felidae. Significant discoveries include the finding that standard cooling techniques for cat sperm result in extensive cell membrane damage, allowing the creation of slower, more effective cooling procedures. Sperm from males producing many malformed cells are less likely to survive cooling-freezing-thawing stress. Egg freezing studies reveal that cat eggs are highly sensitive to cool temperatures. Investigations also continue on the transmission of FIV (related to HIV) in cat semen. A related challenge is the high incidence of abnormally shaped sperm found in the semen of some domestic cats and many endangered cat species. This condition, known as teratospermia and common in men, limits fertilization capacity. Although sperm from teratospermic cats were found to have the same amount of DNA as normal sperm, the former have decreased amounts of protamine, a class of nuclear proteins that play an important role in DNA stabilization. Cats have the short generation times and large litter sizes required for development of transgenic and knock-out research models. Successful adaptation of these techniques to the cat would greatly increase the ability of researchers to develop feline models of human genetic diseases. In collaboration with James Kehler (University of Pennsylvania, School of Veterinary Medicine), we are determining the conditions necessary to propagate and to genetically manipulate feline embryos and to establish feline embryonic stem (ES) and embryonic germ (EG) cells. Transgenic feline embryos will be generated through pronuclear injection and RNA interference. Retroviral vectors will be used to transduce feline primordial germ cells for gain and loss of function experiments. Initial studies will determine whether expression of the transcription factor Oct4 is required for the maintenance of pluripotency in the pre-implantation cat embryo and for formation of feline ES cells, and will determine if Oct4 is required for the survival of cat primordial germ cells and the formation of EG cells. While in mice the transcription factor Oct4 plays a role in maintenance of pluripotency in stem cells and in the survival of germ cells, the role of Oct4 in development in other mammals has not been established. We are developing the cat as a comparative model system for studying the role of Oct4 during mammalian development. Several key resources have been established during the past year to facilitate these developmental studies using cat models. Specifically, a cat colony and gamete laboratory were recently restablished at the NIHAC in Dickerson, MD. A micro-injection station was installed to be able to genetically manipulate cat embryos produced through IVF. Semen has also been collected and cryopreserved from several wildtype and disease cat models for IVF studies.